Methods to differentiate stem cells into hormone-producing cells

ABSTRACT

Methods are described for differentiating stem and post-natal cells into sex hormone-producing cells that can be administered to a patient autologously or allogeneically in order to maintain in balance, or rebalance, their hypothalamic-pituitary-gonadal (HPG) axis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional application and claims priority to U.S.patent application Ser. No. 15/947,304, filed on Apr. 6, 2018, titled“METHODS TO DIFFERENTIATE STEM CELLS INTO HORMONE-PRODUCING CELLS,”which is a Continuation-in-Part application and claims priority to U.S.patent application Ser. No. 14/718,390, filed on May 21, 2015, now U.S.Pat. No. 11,253,549, issued Feb. 22, 2022, titled “METHODS TO REBALANCETHE HYPOTHALAMIC-PITUITARY-GONADAL AXIS,” which claims priority to U.S.Provisional Application No. 62/002,305, filed on May 23, 2014, titled“METHODS TO REBALANCE THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS:APPLICATIONS IN DELAYING AGE-RELATED DISEASES AND EXTENSION OFLONGEVITY,” the disclosures of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to drugs and methods to differentiate stemcells into sex hormone-producing cells. More particularly, thedisclosure relates to specific compounds and combinations of thesecompounds for improving sex hormone production from cells differentiatedusing such treatments.

BACKGROUND OF THE INVENTION

Stem cells have the potential to self-renew (divide) and differentiateinto specialized cell types.

Mesenchymal stem cells (MSC) are a group of clonogenic cells capable ofself-renewal and differentiation displaying phenotypic characteristicsof multilineage mesoderm-type cells, such as osteoblasts, adipocytes,and chondrocytes. Under defined in vitro conditions, MSC have thecapacity to differentiate into ectodermal and endodermal-type cellularlineages. In mammals, MSC have been isolated from many tissue sourcessuch as bone marrow, adipose tissue, skin, cardiac muscle, skeletalmuscle, umbilical cord blood, liver, lung, nasal septum, synovialmembrane, and amniotic membrane.

Several studies have reported protocols to differentiate stem cells intoLeydig-like cells (Miyamoto, et al. 2011; Yazawa, et al. 2016; Yazawa,et al. 2011; Yazawa, et al. 2006).

Studies also have reported protocols to differentiate post-natal cellssuch as fibroblasts into Leydig-like cells (Hou, et al. 2018).

These methods involve stimulation via developmental transcriptionfactors, including the nuclear receptor 5A subfamily (NR5A)proteins—steroidogenic factor-1 (SF-1) and/or liver-specific receptorhomologue-1 (LRH-1), GATA4 and/or NGFI-B together with cAMP treatment(Hou et al. 2018; Miyamoto et al. 2011; Yazawa et al. 2016; Yazawa, etal. 2009; Yazawa et al. 2011; Yazawa et al. 2006).

Injection of such cells can increase circulating sex steroidconcentrations in rodents (Yang, et al. 2017; Yang, et al. 2015; Yazawaet al. 2016).

Wilms tumor gene, Wt1, is abundantly expressed in testis Sertoli cells.Wt1 is required for the lineage specification of both Sertoli andgranulosa cells by repressing Sf1 expression (Chen, et al. 2017).Developmentally, if Wt1 expression is suppressed, the expression of Sf1drives somatic cells to differentiate into steroidogenic cells insteadof supporting cells (Chen et al. 2017). However, SF-1 is essential forSertoli cell maturation and spermatogenesis, during post-natal testisdevelopment (Kato, et al. 2012).

Deletion of the Wt1 results in defects in testosterone biosynthesis,perhaps via a downregulation in the expression of luteinizing hormonereceptor (LHR) on Leydig cells and desert hedgehog (Dhh) expression inSertoli cells (Chen, et al. 2014).

Fetal Leydig cells synthesize only androstenedione as they lack Hsd17b3expression. Fetal Sertoli cells convert androstenedione to testosterone,whereas adult Leydig cells synthesize testosterone by themselves (Shima,et al. 2013).

SUMMARY OF THE INVENTION

The inventors have discovered compounds that differentiate stem cellsinto hormone-producing stem cells (FIG. 1 ). These compounds induce theproduction of sex steroids, including progesterone, 17β-estradiol, andtestosterone.

Namely, the present invention is a method for differentiating stem cellsinto multiple linages including Leydig-like and Sertoli-like cells(hormone-producing cells), comprising stimulating the stem cells via aNR5A transcription factor and WT-1 expression. Furthermore, said methodmay comprise further stimulating the MSC by cAMP.

The inventors have discovered cocktails of compounds that result inmarkedly improved hormone production from stem cells differentiated withthese compounds.

The stem cells can be MSCs derived from bone marrow of fat, or inducedpluripotent stem cells.

The present invention is a method for producing hormone-producing cells,comprising generating hormone-producing cells by implementing saidmethod in vitro.

Moreover, the present invention is for stem cells obtained from humansor other animals.

Embodiments of the present disclosure are capable of utilizing saidcells for balancing and maintaining in balance thehypothalamic-pituitary-gonadal (HPG) axis, at least to some extent.

An embodiment of the invention pertains to a method of treating apatient. In this method, a HPG axis of the patient in need thereof isrebalanced by administering a therapeutically effective amount ofhormone-producing cells.

Another embodiment of the invention relates to a method of reducingendocrine dyscrasia (dyosis) in a patient. In this method, a HPG axis ofthe patient in need thereof is rebalanced by administering atherapeutically effective amount of hormone-producing cells.

Yet another embodiment of the invention pertains to a method of reducingrejection in a patient in need of a tissue-specific stem celltransplant. In this method, a HPG axis of the patient in need thereof isrebalanced by administering a therapeutically effective amount of ahormone-producing cell and administering a second stem cell that istissue-specific to the patient.

Yet another embodiment of the invention relates to a method ofpreventing or slowing dyosis in a patient. In this method, atherapeutically effective amount of at least one physiological agentthat regulates or increases the production of hormones produced by thegonads is administered to a patient.

There has thus been outlined, rather broadly, certain embodiments of theinvention in order that the detailed description thereof herein may bebetter understood, and in order that the present contribution to the artmay be better appreciated. There are, of course, additional embodimentsof the invention that will be described below and which will form thesubject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of embodiments inaddition to those described and of being practiced and carried out invarious ways. Also, it is to be understood that the phraseology andterminology employed herein, as well as the abstract, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

ADVANTAGES OF THE PRESENT INVENTION

The present invention provides an improved method for rapidlydifferentiating stem cells into hormone-producing cells.

The present invention also provides a significant improvement inproduction of multiple sex hormones from stem cells.

These methods allow for the production of more potent stem cells fortransplantation into the gonads of a patient to restore HPG axisbalance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Treatment of rat MSCs with SF-1 agonists promotes progesteroneproduction. Rat MSC's pre-treated for 7 days with RJW100 (1, 5, 10 or 20μM); PE (20, 100 or 200 μM); E2 (100 or 400 nM); DPN (2, 10 or 20 μM);4-HP (50, 200 or 400 nM); RSV (0.44, 1.76 or 3.52 μM), followed bytreatment with N6, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphatesodium (dbcAMP) for 3 days, increases progesterone production.

FIG. 2 : Treatment of rat MSCs with SF-1 agonists promotes testosteroneproduction. Rat MSC's pre-treated for 7 days with RJW100 (1, 5, 10 or 20μM); PE (20, 100 or 200 μM); E2 (100 or 400 nM); DPN (2, 10 or 20 μM);4-HP (50, 200 or 400 nM); RSV (0.44, 1.76 or 3.52 followed by treatmentwith N6, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium(dbcAMP) for 3 days, increases testosterone production. No increase wasobserved for the WT1 inverse agonist 4-HP.

FIG. 3 : Treatment of rat MSCs with SF-1 agonists promotes testosteroneproduction. Rat MSC's pre-treated for 7 days with RJW100 (1, 5, 10 or 20μM); PE (20, 100 or 200 μM); DPN (2, 10 or 20 μM); 4-HP (50, 200 or 400nM); RSV (0.44, 1.76 or 3.52 followed by treatment with N6,2′-O-dibutyryladenosine 3′, 5′-cyclic monophosphate sodium (dbcAMP) for3 days, increases 17β-estradiol production.

FIG. 4 : Treatment of rat MSCs with a combination of SF-1 agonists andthe WT-1 inverse agonist dramatically increases testosterone production.Rat MSC's pre-treated for 7 days with 1) RJW100 (5 μM) PE (20 μM); E2(400 nM), 4) DPN (10 μM) 4-HP (200 nM), or 6) RSV (3.52 μM), or 7)combination of these compounds, followed by treatment with dbcAMP for 3days, increases testosterone production, with the exception of the WT1inverse agonist 4-HP alone.

FIG. 5 : Treatment of human MSCs with an SF-1 agonist increasestestosterone production. Human MSC's pre-treated for 7 days with RJW100(1, 5 and 10 followed by treatment with dbcAMP for 3 days, increasestestosterone production.

FIG. 6 : Treatment of human iCell® MSCs (Cellular DynamicsInternational, Madison, Wis.) with SF-1 agonists and a WT-1 inverseagonist promotes testosterone production. Human MSCs pre-treated for 7days with specific combinations of 1) RJW100 (5 μM); 2) PE (20 3); E2(400 nM), 4) DPN (10 μM) 4-HP (200 nM), and 6) RSV (3.52 μM), followedby treatment with N6, 2′-O-dibutyryladenosine 3′, 5′-cyclicmonophosphate sodium (dbcAMP) for 3 days, increases testosteroneproduction.

FIG. 7 : Injection of educated MSCs into the rat testicle. Rat MSCspretreated for 7 days with the combination of RJW100 (5 PE (20 E2 (400nM), DPN (10 μM), 4-HP (200 nM) and RSV (3.52 μM) were injected into thetestes (1 million cells/testicle) to increase circulating testosteroneconcentrations in 8.5 month old male rats.

The drawings presented are intended solely for the purpose ofillustration and therefore, are neither desired nor intended to limitthe subject matter of the disclosure to any or all of the exact detailsof construction shown, except insofar as they may be deemed essential tothe claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure relates to a method of differentiating stem andadult cells into hormone-producing cells (or ‘steroidogenic cells’; SCs)for use in maintaining in balance or rebalancing, the HPG axis andpreventing or reversing hypogonadism and accompanying symptoms anddiseases.

The transcriptional factor SF-1 is an inducing factor that is stimulatedor blocked by various agonists, inverse agonists and antagonists in thepresent invention, is an orphan intranuclear receptor, which isexpressed in genital and adrenal gland-type steroid hormone-producingcells, and has been known to control the transcription of steroidhormone producing-enzymes (Morohashi and Omura 1996; Parker and Schimmer1997). Even if SF-1 is derived from different animal species, SF-1 bindsto a common target DNA sequence in mesenchymal stem cells and the factoris expected to provide the same result.

The transcriptional factor Wilm's tumor protein (WT-1), is an inducingfactor (Barrionuevo, et al. 2012; Chen et al. 2017) that is stimulatedor blocked by various agonists, inverse agonists and antagonists in thepresent invention.

cAMP exists ubiquitously in all living organism, whose intra-cellularconcentration is 10⁻⁶ to 10⁻⁷ M. cAMP participates in generation ofspecific enzymes and metabolic control in target cells and alsoparticipates in growth and differentiation of cells. cAMP is a secondmessenger of luteinizing hormone (LH) and adrenocorticotropic hormone(ACTH), which induces the expression of steroid hormoneproduction-related enzymes in genital and adrenal glands and enhancesthe production of steroid hormones.

Stimulation of these transcription factors (SF1 and WT1) by cAMP mayinclude the direct contact of these factors with cells or the use of avector expressing these factors.

To differentiate stem cells into hormone-producing cells, stem cells maybe stimulated by an inducing factor in vitro. For example, mesenchymalstem cells may be cultured in a media containing serum or serumcomponents in an incubator with 5% CO₂ at 37° C. (see examples).

To differentiate adult cells into hormone-producing cells, adult cellsmay be stimulated by an inducing factor in vitro. For example,fibroblasts may be cultured in a media containing serum or serumcomponents in an incubator with 5% CO₂ at 37° C. (see examples).

The method involves treating stem or adult cells cultured in appropriatemedias with one of the following:

-   -   1. RJW100 ((Whitby, et al. 2006; Whitby, et al. 2011); LRH-1,        SF-1 agonist)    -   2. Phenylephrine (PE; (Favaretto, et al. 1988; Mayerhofer, et        al. 1989); enhances hCG-mediated T secretion, alpha-adrenergic        agonist)    -   3. 17β-Estradiol (E2; (Kumar, et al. 2016); promotes Sertoli        cell proliferation)    -   4. 2,3-Bis(4-hydroxyphenyl)propionitrile ((Royer, et al. 2012;        Sato, et al. 2018); DPN)    -   5. 4-(Heptyloxy)phenol (4-HP; (Del Tredici, et al. 2008); SF-1        inverse agonist)    -   6. Resveratrol (RSV; (Wu, et al. 2012); SF-1 agonist)    -   7. Dimethyl sulfoxide (DMSO)

Treatment of stem cells (or iPS cells) individually for 4-7 days withthe following compounds at the following concentrations: RJW100 (104, 5μM, 10 μM, 20 μM); PE (20 μM, 100 μM, 200 μM); E2 (100 nM, 400 nM); DPN(2 μM, 10 μM, 20 μM); 4-HP (50 nM, 200 nM, 400 nM); RSV (0.44, 1.76 or3.52 μM), followed by treatment with N6, 2′-O-dibutyryladenosine3′,5′-cyclic monophosphate sodium (dbcAMP) for 3 days increasesprogesterone, testosterone and 17β-estradiol production (FIGS. 1-6 ).

An improved method involves treating stem cells (or iPS cells) culturedin appropriate medias with more than one of the following RJW100 (5 μM),PE (20 μM); E2 (400 nM); DPN (10 μM); 4-HP (200 nM), and RSV (3.52 μM).In particular, the addition of the SF-1 agonist (4-HP), that promotesSertoli cell differentiation by repressing Sf1 expression (Chen et al.,2017), markedly increases testosterone production from bone-derived MSCand human iCell® MSCs (Cellular Dynamics International, Madison, Wis.;FIGS. 4 and 6 ).

The presence of Sertoli cells in these cultures provides factors thatpromote steroid production from neighboring Leydig-like cells.

When this mixture of hormone-producing cells were transplanted into amammalian reproductive organ, fat pad, or intravenously, there was anincrease in circulating testosterone, progesterone and estradiol (FIG. 7).

When hormone-producing cells were transplanted into a mammalianreproductive organ, certain factors activate the cell (endogenous cAMP,LH, FSH) to drive sex hormone production and increased circulatinghormone concentrations.

A variation to this method is to inject the transplanted patient withgonadotropin (hCG, LH and/or FSH) at doses sufficient to induce hormoneproduction from the transplanted cells.

When hormone-producing cells were transplanted into a mammalianreproductive organ, certain factors further differentiate the cell(endogenous cAMP, LH, FSH) to become Leydig, Sertoli and other gonadalcells.

Since cAMP is necessarily present in all cells with a large variety ofconcentrations, intracellular concentration of cAMP will act ontransplanted stem cells without adding exogenous cAMP.

Hormone-producing cells derived from stem cells include testicularLeydig cells, testicular Sertoli cells, testicular macrophages, ovariangranulosa cells, ovarian capsular cells, ovarian thecal cells, ovarianmacrophages, adrenal cortical cells, and others.

Embodiments of the present invention relates to a method for slowing,preventing, or delaying senescence, preventing, or treating a diseaseassociated with senescence, and for increasing longevity. This isachieved by delivering donor cells into the human or animal body toincrease the production and secretion of sex hormones into thecirculation to levels near young adult reproductive levels, therebyreinitiating negative feedback on the hypothalamus and pituitary torebalance the HPG axis hormone synthesis and secretion to levels nearyoung adult reproductive levels. This in effect prevents dyotic (death)signaling that results from the dysregulation of the HPG axis (Atwoodand Bowen 2011; Atwood, et al. 2017; Bowen and Atwood 2004). This willprevent and treat hypogonadism, prevent, and treat symptoms associatedwith female reproductive endocrine dyscrasia and symptoms associatedwith male reproductive endocrine dyscrasia, and prevent or delay theonset of age-related diseases and extend longevity.

The invention encompasses a method of preventing or reversing thedysregulation of the HPG axis by repopulating the ovaries withfollicular cells, and the testes with Leydig, Sertoli and other supportcells. This will prevent and treat hypogonadism, prevent, and treatsymptoms associated with female reproductive endocrine dyscrasia andsymptoms associated with male reproductive endocrine dyscrasia, andprevent and delay the onset of age-related diseases and extendlongevity.

The invention encompasses a method of restoring the HPG axis to balance(young adult reproductive levels) by repopulating the ovaries withfollicular cells, and the testes with Leydig, Sertoli and other supportcells. This will reverse hypogonadism, prevent and treat symptomsassociated with female reproductive endocrine dyscrasia and symptomsassociated with male reproductive endocrine dyscrasia, and prevent anddelay the onset of age-related diseases and extend longevity.

The invention further encompasses a method of inhibiting inflammationsuch as decreasing the expression of tumor necrosis factor (TNF), in asubject, by administering donor cells that lead to a rebalancing of theHPG axis.

Thus, the present invention encompasses reversing the degenerative serumhormonal milieu back to one that allows the appropriate growth anddevelopment of cells for the normal maintenance of tissue structure andfunction in the body. Rebalancing the endocrine HPG axis will allow forthe rebalancing of the tissue specific ‘mini-HPG’ axes present intissues throughout the body (Meethal et al. 2009b). This will rebalancereproductive hormone signaling to cells in all tissues of the body.

The present invention encompasses a method of maintaining HPG axishormones in balance to extend longevity for purposes of extendinglongevity of animals with agricultural applications, such as increasingyields of wool, cashmere or other fibers per animal. Similarapplications apply to egg and milk production.

This can be achieved by injecting into a subject donor cells that canrepopulate the gonads with cell types capable of producing reproductivehormones required to balance the HPG axis. For male subject, donor cellscapable of differentiating into germ cells (spermatogonia,spermatocytes, spermatids and spermatozoon), Sertoli cells, myoid cells,Leydig cells, stromal cells, macrophage cells and/or epithelial cellsand integrating into the tissue to restore function. For female subject,donor cells capable of differentiating into germ cells (oogonial stemcells), granulosa cells, cumulus cells, thecal cells, stromal cells,epithelial cells, macrophage cells and/or oocyte cells, and integratinginto the tissue to restore function.

The differentiation of donor cells into more than one gonadal cell typeis required to allow complete rebalancing of the axis. For example,while Leydig cells primarily produce androgens, Sertoli cells producelarge quantities of inhibins, both of which are required for HPG axisrebalancing in males. A combination of gonadal cells is optimal forcomplete rebalancing of the axis.

An embodiment of the present invention includes administering, to asubject, donor cells that decrease or regulate the blood levels,production, function or activity of gonadal hormone to be near the bloodlevels, production, function or activity occurring during fetal life orat or around the height of the subject's reproductive period, which inhumans usually corresponds to about 18 to 35 years of age.

In another embodiment, the present invention encompasses administering,to a subject, donor cells that decrease or regulate the blood levels,production, function or activity of kisspeptin, GnRH, LH or FSH to beapproximately as low as possible without significant adverse sideeffects, preferably to be undetectable or nearly undetectable byconventional detection techniques known in the art, which, at thepresent time, is less than 0.7 mIU/mL for both LH and FSH. In anotherembodiment, the present invention encompasses administering, to asubject, donor cells that regulate the function or activity of activinto be approximately as low as possible without significant adverse sideeffects, preferably to be undetectable or nearly undetectable byconventional detection techniques known in the art. In anotherembodiment, the present invention encompasses administering donor cellsthat increase or regulate the blood levels, production, function, oractivity of inhibin, follistatin, myostatin or BMP4 to be approximatelyas high as possible without significant adverse side effects.

In other embodiments of the present invention, the blood levels,production, function, or activity of gonadal hormones are continuouslyregulated, by monitoring the blood levels, production, function, oractivity and making adjustments to the donor cell or donor cells beingadministered via a feedback control system.

Embodiments of the present invention include administration of one ormore stem or differentiated cell types that can be used to increase orregulate the blood and/or tissue levels, production, function, oractivity of gonadal hormones. Studies have shown that increasing thelevels of circulating sex steroids and inhibins will result insignificant decreases in GnRH, LH and FSH levels and a rebalancing ofthe HPG axis (Hayes, et al. 1998; Thorner et al. 1998; Ying 1988).Through a negative feedback loop, the presence of sex steroid hormonessuch as estrogen, testosterone or progesterone signals the hypothalamusto decrease the secretion of GnRH (Gharib, et al. 1990; Steiner, et al.1982). The subsequent decrease in GnRH decreases the secretion of LH andFSH (Thorner et al. 1998). For example, sex steroids, inhibins andfollistatin have been shown to provide negative feedback regulation ofGnRH and FSH synthesis and secretion (Bagatell, et al. 1994; Boepple, etal. 2008; Dubey, et al. 1987; Hayes, et al. 2001b; Illingworth, et al.1996; Lambert-Messerlian, et al. 1994; Marynick, et al. 1979; Pitteloud,et al. 2008a, b; Schnorr, et al. 2001; Sherins and Loriaux 1973;Winters, et al. 1979a; Winters, et al. 1979b) while sex steroids appearto primarily provide negative feedback for the regulation of GnRH and LHsynthesis and secretion (Bagatell et al. 1994; Hayes, et al. 2001a;Santen 1975; Schnorr et al. 2001; Veldhuis, et al. 1992). In females,sex steroids, inhibins and follistatin have been shown to providenegative feedback regulation of FSH (le Nestour, et al. 1993; Welt, etal. 1997) and LH (Jaffe and Keye 1974, 1975; Jaffe, et al. 1976; Keyeand Jaffe 1974, 1975, 1976; Liu and Yen 1983; Taylor, et al. 1995; Youngand Jaffe 1976) synthesis and secretion.

Embodiments of the present invention also encompass rebalancing of theHPG axis such that the axis and related hormonal concentrations arebalanced for that person. The production of sex hormones by donor cellsis expected to be different for different individuals in order to reachoptimal balancing of that person's HPG axis. Thus, the circulating andtissue concentrations of sex hormones in one person's balanced HPG axisis expected to be different to that of another person whose axis is alsobalanced.

Embodiments of the present invention also encompass theminute-to-minute, hour-to-hour and day-to-day variations in HPG axishormone production to allow the axis to remain in balance.

Embodiments of the present invention also encompass modulating theconcentrations and ratios of hormones of the HPG axis at any stage ofthe life cycle, including the embryo, fetus, childhood, puberty,adulthood or during senescence.

Embodiments of the present invention also encompass modulating theconcentrations and ratios of hormones of the HPG axis during gendertransition from male to female, or female to male.

Embodiments of the present invention also encompass returning the ratiosof sex hormones back to near the ratios occurring during fetal life orat or near the time of greatest reproductive function of the subject.For example, the ratio of testosterone:FSH during the male reproductiveperiod is ˜11 (6.5 ng/mL:0.6 ng/mL), while that during thepost-reproductive period (post-menopause) is ˜1 (2.3 ng/mL:2.3 ng/mL).In this example, treatment would aim to return the ratio of thesehormones back to 11. Further embodiments to this invention wouldencompass returning all the sex hormone ratios back to those duringfetal life or at the time of greatest reproductive function of thesubject.

Embodiments of the present invention also encompass administration ofpurified and mixed donor cell populations derived from the tissues of anindividual who will receive the donor cells.

Embodiments of the present invention also encompass administration to anindividual purified and mixed donor cell populations derived frommultiple tissues of one or more individuals.

Embodiments of the present invention encompass administration ofautologous or allogenic donor cell populations into the gonads for theprevention or treatment of hypogonadism, hypergonadotropic hypogonadism,andropause, menopause and related conditions, and for the prevention andtreatment of diseases associated with senescence and aging.

Embodiments of the present invention also encompass administration ofdonor cell populations into the gonads prior to administration of donorcell populations (e.g. stem cell therapy, iPS therapy, orimplantation/injection of differentiated cells including stem cells thathave been differentiated in vitro) into other tissues of the body. Sucha method allows for rebalancing the HPG axis so that the ‘toxicenvironment of dyotic signaling’ is reversed in order to allow for donorcells transplanted into other tissues to differentiate appropriately,integrate into the tissue and restore function.

In other embodiments of the invention, donor cell recipients may receivesupplemental gonadal hormones, GnRH agonists/antagonists, anLH/FSH-inhibiting agent, an activin-inhibiting agent, aninhibin-promoting agent, and/or a follistatin-promoting agent.

According to embodiments of the present invention, administration ofGnRH agonists/antagonists, LH/FSH-inhibiting agents,activin-inhibiting-agents, inhibin-promoting agents,follistatin-promoting agents, or sex steroids, including those listedabove, can be oral or by injection, inhalation, patch, or othereffective means. According to embodiments of the present invention, thedosage of GnRH agonists/antagonists, LH/FSH-inhibiting agents,activin-inhibiting agents, inhibin-promoting agents,follistatin-promoting agents, or sex steroids, including thoseidentified above, will be a therapeutically effective amount, sufficientto decrease or regulate the blood and/or tissue levels, production,function or activity of GnRH, LH or FSH, or to decrease or regulate thefunction or activity of activin or to increase or regulate the bloodand/or tissue levels, production, function or activity of inhibin orfollistatin, to the desired blood and/or tissue levels, production,function or activity. According to other embodiments of the invention,administration of LH/FSH-inhibiting agents, activin-inhibiting agents,inhibin-promoting agents, follistatin-promoting agents, or sex steroids,including those identified above, can be in a single dose, multipledoses, in a sustained release dosage form, in a pulsatile form, or inany other appropriate dosage form or amount. Administration prior totreatment with cells is preferred, but can occur during or afteradministration of cells. The duration of treatment could range from afew days or weeks to the remainder of the patient's life.

In addition to treating neurodegenerative diseases, the administrationof GnRH agonists/antagonists, LH/FSH-inhibiting agents,activin-inhibiting agents, inhibin-promoting agents,follistatin-promoting agents, sex steroids, or other agents thatdecrease dysregulated cell cycle signaling, as described above, isexpected to be beneficial as a prophylactic or in the treatment of agingand diseases where cell replenishment is required in order to repopulatea tissue to regain function or establish a new function, in accordancewith the present invention.

EXAMPLES Example 1: General Overview of Stem Cell Education intoHormone-Producing Stem Cells

Adult stem mesenchymal stem cells (MSCs) (or bone marrow stromal cells),are pluripotent cells that have the ability to differentiate into cellsof all three germ layers (Ratajczak, et al. 2008).

In the case of a human male or female, MSCs are isolated from 1) bone,the femur and/or tibia (Tuli, et al. 2003a; Tuli, et al. 2003b), 2)umbilical cord blood (Hayward, et al. 2013; Malgieri, et al. 2010), 3)Wharton's jelly (Hayward et al. 2013), 4) skin (Manini, et al. 2011) or5) adipose tissue (Kuhbier, et al. 2010; Manini et al. 2011; Tholpady,et al. 2003; Zhu, et al. 2013; Zuk, et al. 2001). Cells are thensubjected to flow cytometry to isolate MSC that are then injected(10,000-1 billion cells/treatment) into the interstitium of one or bothtestes or ovaries of the donor. In the testes, cells can be injectedinto the seminal vesicle lobules, septa, tunica albuginea, straighttubule, rete testes, efferent ductile and/or epididymis. In the ovary,cells can be injected into the ovarian cortex. If the number of isolatedMSCs is insufficient, MSCs are expanded in culture first prior toinjection into the gonads.

Stem cells and educated stem cells (e.g. MSCs differentiated intohormone-producing stem cells) injected into the testes localize to thetesticular interstitium and seminiferous tubules and differentiate intoLeydig cells and spermatogonia/spermatocytes, respectively (Lo, et al.2004; Yazawa et al. 2006). Stem cells injected into the ovaries increasefollicle numbers (Abd-Allah, et al. 2013). Hormonal factors secretedwithin the gonads direct the differentiation and integration of suchstem cells for the replenishment of germ cells, Leydig, Sertoli andother cells in the testes, and replenishment of follicular cells (germcells, granulosa, thecal and other cells) in the ovaries. Hormonessecreted by the transplanted cells and their progeny in turn rebalancethe HPG axis.

This technique may be performed autologously, i.e. isolating cells fromthe same individual who will receive the cells; allogeneically, i.e.cells isolated from one individual are injected into another individual(human or animal); or both autologously and allogeneically, i.e.isolated cells from the recipient and from another individual(s) areinjected into the recipient.

In this example, MCSs, from which gonadal tissues are derived duringembryogenesis, are purified from tissues other than the gonads and theninjected into the gonads.

MSCs in a suitable buffer, or encapsulated in a hydrogel or other matrix(e.g. fibrin, collagen) prior to injection, may be injected into thegonads (testes or ovaries). Injection may be via a catheter. MSCs inthis example are capable of differentiating into all relevant gonadalcell types upon injection into the gonads.

This technique can be used on humans, animals and plants with areproductive hormone axis.

Following transplantation of cells, the patient can be treated withgonadotropins (hCG, LH and/or FSH) to induce further differentiationhormone production.

The concentration of circulating reproductive hormones in the individualcan be measured before and after the injection of cells to confirm thatinjected cells are producing hormones and rebalancing the HPG axis.Tissue concentrations of reproductive hormones can be measured intissues to confirm that the hormones of the ‘mini-HPG-axis in thattissue have rebalanced (returned to young adult reproductiveconcentrations). If the HPG axis has not completely rebalanced, a secondor subsequent injection can be given until such time as the HPG axis isbalanced and dyotic signaling has decreased. This provides apreventative and treatment for hypogonadism (primary) and of age-relatedreproductive endocrine dyscrasia.

Example 2: MSC Education into Hormone-Producing Cells

MSCs or other stem cell populations are differentiated in vitro intodiscrete precursor or differentiated cell types including germ cells(spermatogonia, spermatocytes, spermatids and spermatozoon), Sertolicells, myoid cells, Leydig cells, stromal cells, macrophage cells and/orepithelial cells in the case of the male; or germ cells (oogonial stemcells), granulosa cells, cumulus cells, thecal cells, stromal cells,epithelial cells, macrophage cells and/or oocyte cells, in the case ofthe female, and one or preferably more of these cell types are injectedinto the gonads and/or other tissues and circulating and tissue sexhormone concentrations measured as in the methods described in Example1, for rebalancing of the HPG axis.

Bone marrow derived hMSC are grown to confluence in T75 flasks usingMinimum Essential Medium (MEM)—Alpha 1, with Earle's salts, GlutagroSupplement, L-alanyl-L-glutamine, MEM nonessential amino acids andHyClone fetal bovine serum prior to treatment with differentiationfactors.

MSCs are differentiated into hormone-producing cells over 7-21 days in awater-jacketed CO₂ incubator (Thermo Electron Corporation, Waltham,Mass.) at 37° C. with 5% CO₂ by treatment with RJW100 (5 μM), PE (20μM); E2 (400 nM); DPN (10 μM); 4-HP (200 nM), and RSV (3.52 μM).

Hormone production can be induced in educated cells by treatment withN6, 2′-O-dibutyryladenosine 3′, 5′-cyclic monophosphate sodium (dbcAMP),or following treatment with LH, FSH and/or hCG, for 1-3 days in serumsupplement-free media.

Sex steroid and protein hormone concentrations are measured in themedia.

Cells are then injected into a patient, including the gonads or fat pad,and the patient can be treated with gonadotropins (LH, FSH and/or hCG)to aid in repopulation, cell differentiation and hormone production.

Example 3

Induced pluripotent stem (iPS) cells created from the recipient oranother donor can be cultured to produce sufficient cell numbers to beinjected into either one or both of the gonads, and/or injected into thecirculation, and/or other tissues of the body and circulating and tissuesex hormone concentrations measured as described in Example 1 torebalance the HPG axis. Differentiated cells such as fibroblasts,umbilical cord fibroblasts stomach, hepatocytes, lymphocytes, prostaticcells and other adult differentiated cells can be obtained by varioustechniques known in the field and reprogrammed into iPS cells via thefollowing techniques also known in the field.

Human iCell® Mesenchymal Stem Cells (iCell MSC; Cellular DynamicsInternational, Madison, Wis.) are grown to confluence in iCellmaintenance media that includes L-Ascorbic Acid, B-27 supplement minusvitamin A, recombinant human FGF-basic, bovine serum albumin, GlutaMAXsupplement, Ham's F-12 medium, Iscove's Modified Dulbecco's Medium,1-Thioglycerol, N-2 supplement, recombinant human PDGF-BB, andpenicillin/streptomycin. The iCell MSC are differentiated intohormone-producing cells over 7-21 days in a water-jacketed CO₂ incubator(Thermo Electron Corporation, Waltham, Mass.) at 37° C. with 5% CO₂ bytreatment with RJW100 (5 μM), PE (20 μM); E2 (400 nM); DPN (10 μM); 4-HP(200 nM), and RSV (3.52 μM).

Hormone production can be induced in educated cells by treatment withN6, 2′-O-dibutyryladenosine 3′, 5′-cyclic monophosphate sodium (dbcAMP),or following treatment with LH, FSH and/or hCG, for 1-3 days in serumsupplement-free media.

Sex steroid and protein hormone concentrations are measured in themedia.

Cells can be administered to patients as described in the examples aboveand below.

Example 4

The above techniques can also be used to differentiate post-natalfibroblasts from foreskin or punch biopsies into Leydig-like andSertoli-like cells.

Example 5

Adult testicular cells such as Leydig cells, Sertoli cells, and germcells can be differentiated from MSCs following transfection withmembers of the nuclear receptor family, SF-1, liver receptor homolog-1(LRH-1), and/or Wilms tumor protein (WT1), and treatment with8-bromoadenosine-cAMP ((Yazawa et al. 2006); WT1). One or preferablyboth of these cell types are injected into the male gonads and/or othertissues neat or in matrices via methods described in Example 1 andcirculating and tissue sex hormone concentrations measured as in themethods described in Example 1, for rebalancing of the HPG axis. Cellsmay be autologous or allogeneic. In a derivation of this method, MSC orother cell types are treated with differentiation factors as describedin Example 3, and injected within 24 h into the testes via methodsdescribed in Example 1. In another derivation of this method, MSC orother cell types are imbedded in a matrix impregnated withdifferentiation factors and injected into the testes via methodsdescribed in Example 1.

Generation of iPSCsReprogramming with Lentiviral Transduction

Three plasmid vectors of lentiviral reprogramming: FUW-tetO-lox-hO2S,FUW-tetO-lox-hM2K, and FUW-tetO-lox-hN2L are constructed. Expressioncassettes of human POU5F1-internal ribosome entry site 2 (IRES2)-SOX2(O2S) and MYC-IRES2-KLF4 (M2K) of pEP4 EO2S EM2K (Addgene, #20923) (Yu,et al. 2009) are used for the O2S and M2K cassettes. Pseudovirus isproduced in 293FT cells by transfection with each lentiviral vector(O2S, M2K, N2L) and the reverse tetracycline transactivator expressionplasmid, FUW-M2rtTA (Addgene, plasmid 20342) (Hockemeyer, et al. 2008)along with the VSV-G envelope (pMD2.G) and packaging vector (psPAX2)(Ezashi, et al. 2009). Two consecutive infections are introduced intothe target cell or interest (1×10⁵ cells) in the presence of 12 μg/mlhexadimethrine bromide (polybrene, Sigma, St. Louis, Mo.). During theinfection stage, the cells are cultured for 48 h by adding a mixture ofthe four titered pseudoviruses (multiplicity of infection); O2S (30.8),M2K (17.5), N2L (18.2) and rtTA (20.7) to the culture medium. On day 4after infection, cells are dispersed with trypsin and then expanded.Cells are tested for pluripotency and can then be used for treatment.

Reprogramming with Episomal Plasmids

Episomal vectors carrying the reprogramming genes SOX2, KLF4, POU5F1,LIN28, p53 and MYCL (combined episomal plasmids; Addgene #27077, 27078and 27080) are electroporated into 1-6×10⁵ cells using a Nucleofector IIdevice (Lonza, Basel, Switzerland) and Amaxa NHDF Nucleofector kit(Lonza). After 20 days, colonies resembling human ESC are mechanicallyisolated and expanded in mTeSR1 medium (Gallego, et al. 2010; Ludwig, etal. 2006; Porayette, et al. 2009) (StemCell Technologies, Vancouver,Canada) on a Matrigel (BD Bioscience, San Jose, Calif.) coatedsubstratum. Cells are tested for pluripotency and can then be used fortreatment.

Example 6

Adult granulosa, cumulus, thecal and germ cells can be isolated fromadult ovaries following tituration, percoll gradients and/or flowcytometry (Sittadjody, et al. 2013) and one or preferably more of thesecell types, educated cell types, or bioengineered cell types asdescribed in the above examples injected into the female gonads and/orother tissues and circulating and tissue sex hormone concentrationsmeasured as in the methods described in Examples 1-4, for rebalancing ofthe HPG axis.

Example 7

This technique can be used on humans, animals and plants with areproductive hormone axis.

Example 8

The patient is pre-treated with agents to lower dyotic signaling, suchas GnRH agonists/antagonists and/or sex steroid supplementation (e.g.testosterone in males; estradiol and progesterone in females), prior totreatment with donor cells as outlined in Examples 1-7 to aid in therepopulation of gonadal cells.

Pre-treatment of patients described above is performed prior to theinjection of donor cells into non-gonadal tissues or the circulation,and tissue regeneration and function monitored.

Example 9

The above methods in Examples 1-8 can be utilized to rebalance the HPGaxis and reverse or prevent dyotic signaling in tissues, therebyallowing for a more conducive environment for innate tissue regenerationor regeneration aided by treatment with donor cells. The methods fromExamples 1-8 can be performed on patients, circulating and tissue sexhormone concentrations measured to confirm the HPG axis is rebalancedand that dyotic signaling has decreased, prior to the injection of donorcells into specific tissues or the circulation, and tissue regenerationand function monitored. As one example, the method of Example 1 can beused to decrease dyotic signaling to the brain, and donor cells (e.g.neural stem cells, iPS cells or differentiated neural cells) injectedinto a dysfunctioning region(s) of the brain.

Example 10

These techniques can be used to treat hypogonadotropic hypogonadism(secondary hypogonadism), a condition characterized by hypogonadism dueto an impaired secretion of gonadotropins, including FSH and LH, by thepituitary gland in the brain, and in turn decreased gonadotropin levelsand a resultant lack of sex steroid production. Pituitary cell typessuch as gonadotrophs, corticotrophs, thyrotrophs, lactotrophs andadipose generated by way of Examples 1-3, 5-7, and from pituitarytissue, can be cultured to produce sufficient cell numbers to beinjected into the pituitary, and/or injected into the circulation,and/or other tissues of the body to rebalance the HPG axis as describedin Examples 1-8 with or without pre-treatment of patients described inExample 8. Circulating and tissue sex hormone concentrations measured asdescribed in Example 1 are performed to confirm rebalancing of the HPGaxis. Conditions and diseases treated by this method include secondarycongenital forms of hypogonadism (hypogonadotropic hypogonadism):Kallman syndrome, isolated GnRH deficiency, isolated LH deficiency,Prader-Willi syndrome, Turner syndrome, and Laurence-Moon-Biedlsyndrome; and secondary acquired forms of hypogonadism: pituitary tumorsand infarct, trauma, mumps, traumatic brain injury, children born tomothers who had ingested the endocrine disruptor diethylstilbestrol,opioid induced androgen deficiency (resulting from the prolonged use ofopioid class drugs, e.g. morphine, oxycodone, methadone, fentanyl,hydromorphone), anabolic steroid-induced hypogonadism craniopharyngioma,hyperprolactemia (1° &2°), hemochromatosis and neurosarcoid.

Example 11

The above techniques also can be used to treat other dysregulatedhormone axes of the body, including conditions and diseases thatdysregulate the hypothalamic-pituitary-adrenal axis (e.g. adrenalinsufficiency, Cushing's syndrome, Addison disease), alimentary systemhormone axes, placental hormone axes, calcium regulatory axes, saltregulatory axes, thermoregulatory axes and thyroid hormone axes

Example 12

The above techniques in Examples 1-12 can be used to treat animals suchas stud bulls or horses, pets and members of rare and endangered speciesin order to restore hormone balance and improve or maintain health andlifespan.

CONCLUSION

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not by limitation. For example, the present inventionis not limited to the stem or differentiated cells illustrated ordescribed, the methods of injection, the hormones produced by the cells,or the injected tissues illustrated or described. In another example,although some cells and techniques described herein are related tohumans, the present invention is not limited to humans, but rather,includes all reproductively viable organisms. As such, the breadth andscope of the present invention should not be limited to any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

REFERENCES

-   Abd-Allah S H, Shalaby S M, Pasha H F, El-Shal A S, Raafat N,    Shabrawy S M, Awad H A, Amer M G, Gharib M A, El Gendy E A, et al.    2013 Mechanistic action of mesenchymal stem cell injection in the    treatment of chemically induced ovarian failure in rabbits.    Cytotherapy 15 64-75-   Atwood C S & Bowen R L 2011 The reproductive-cell cycle theory of    aging: an update. Experimental Gerontology 46 100-107.-   Atwood C S, Hayashi K, Meethal S V, Gonzales T & Bowen R L 2017 Does    the degree of endocrine dyscrasia post-reproduction dictate    post-reproductive lifespan? Lessons from semelparous and iteroparous    species. GeroScience 39 103-116.-   Barrionuevo F J, Burgos M, Scherer G & Jimenez R 2012 Genes    promoting and disturbing testis development. Histology and    histopathology 27 1361-1383.-   Bowen R L & Atwood C S 2004 Living and dying for sex. A theory of    aging based on the modulation of cell cycle signaling by    reproductive hormones. Gerontology 50 265-290.-   Chen M, Wang X, Wang Y, Zhang L, Xu B, Lv L, Cui X, Li W & Gao F    2014 Wt1 is involved in leydig cell steroid hormone biosynthesis by    regulating paracrine factor expression in mice. Biology of    Reproduction 90 71.-   Chen M, Zhang L, Cui X, Lin X, Li Y, Wang Y, Qin Y, Chen D, Han C,    Zhou B, et al. 2017 Wt1 directs the lineage specification of sertoli    and granulosa cells by repressing Sf1 expression. Development 144    44-53.-   Del Tredici A L, Andersen C B, Currier E A, Ohrmund S R, Fairbain L    C, Lund B W, Nash N, Olsson R & Piu F 2008 Identification of the    first synthetic steroidogenic factor 1 inverse agonists:    pharmacological modulation of steroidogenic enzymes. Molecular    pharmacology 73 900-908.-   Ezashi T, Telugu B P, Alexenko A P, Sachdev S, Sinha S & Roberts R M    2009 Derivation of induced pluripotent stem cells from pig somatic    cells. Proceedings of the National Academy of Sciences of the United    States of America 106 10993-10998.-   Favaretto A L, Valenca M M, Hattori M, Wakabayashi K &    Antunes-Rodrigues J 1988 Characterization of adrenergic control of    the Leydig cell steroidogenesis: identification of both stimulatory    and inhibitory components. Brazilian journal of medical and    biological research=Revista brasileira de pesquisas medicas e    biologicas 21 539-543.-   Gallego M J, Porayette P, Kaltcheva M M, Bowen R L, Vadakkadath    Meethal S & Atwood C S 2010 The pregnancy hormones human chorionic    gonadotropin and progesterone induce human embryonic stem cell    proliferation and differentiation into neuroectodermal rosettes.    Stem cell research & therapy 1 28.-   Hayward C J, Fradette J, Galbraith T, Remy M, Guignard R, Gauvin R,    Germain L & Auger F A 2013 Harvesting the potential of the human    umbilical cord: isolation and characterisation of four cell types    for tissue engineering applications. Cells, tissues, organs 197    37-54.-   Hockemeyer D, Soldner F, Cook E G, Gao Q, Mitalipova M & Jaenisch R    2008 A drug-inducible system for direct reprogramming of human    somatic cells to pluripotency. Cell stem cell 3 346-353.-   Hou Y P, Zhang Z Y, Xing X Y, Zhou J & Sun J 2018 Direct conversion    of human fibroblasts into functional Leydig-like cells by SF-1,    GATA4 and NGFI-B. American journal of translational research 10    175-183.-   Kato T, Esaki M, Matsuzawa A & Ikeda Y 2012 NR5A1 is required for    functional maturation of Sertoli cells during postnatal development.    Reproduction 143 663-672.-   Kuhbier J W, Weyand B, Radtke C, Vogt P M, Kasper C & Reimers K 2010    Isolation, characterization, differentiation, and application of    adipose-derived stem cells. Advances in biochemical    engineering/biotechnology 123 55-105.-   Kumar N, Srivastava S, Burek M, Forster C Y & Roy P 2016 Assessment    of estradiol-induced gene regulation and proliferation in an    immortalized mouse immature Sertoli cell line. Life sciences 148    268-278.-   Lo K C, Lei Z, Rao Ch V, Beck J & Lamb D J 2004 De novo testosterone    production in luteinizing hormone receptor knockout mice after    transplantation of leydig stem cells. Endocrinology 145 4011-4015.-   Ludwig T E, Bergendahl V, Levenstein M E, Yu J, Probasco M D &    Thomson J A 2006 Feeder-independent culture of human embryonic stem    cells. Nature methods 3 637-646.-   Malgieri A, Kantzari E, Patrizi M P & Gambardella S 2010 Bone marrow    and umbilical cord blood human mesenchymal stem cells: state of the    art. International journal of clinical and experimental medicine 3    248-269.-   Manini I, Gulino L, Gava B, Pierantozzi E, Curina C, Rossi D, Brafa    A, D'Aniello C & Sorrentino V 2011 Multi-potent progenitors in    freshly isolated and cultured human mesenchymal stem cells: a    comparison between adipose and dermal tissue. Cell and tissue    research 344 85-95.-   Mayerhofer A, Bartke A & Steger R W 1989 Catecholamine effects on    testicular testosterone production in the gonadally active and the    gonadally regressed adult golden hamster. Biology of Reproduction 40    752-761.-   Miyamoto K, Yazawa T, Mizutani T, Imamichi Y, Kawabe S Y, Kanno M,    Matsumura T, Ju Y & Umezawa A 2011 Stem cell differentiation into    steroidogenic cell lineages by NR5A family. Molecular and cellular    endocrinology 336 123-126.-   Morohashi K I & Omura T 1996 Ad4BP/SF-1, a transcription factor    essential for the transcription of steroidogenic cytochrome P450    genes and for the establishment of the reproductive function. FASEB    journal: official publication of the Federation of American    Societies for Experimental Biology 10 1569-1577.-   Parker K L & Schimmer B P 1997 Steroidogenic factor 1: a key    determinant of endocrine development and function. Endocrine reviews    18 361-377.-   Porayette P, Gallego M J, Kaltcheva M M, Bowen R L, Vadakkadath    Meethal S & Atwood C S 2009 Differential processing of amyloid-beta    precursor protein directs human embryonic stem cell proliferation    and differentiation into neuronal precursor cells. The Journal of    biological chemistry 284 23806-23817.-   Royer C, Lucas T F, Lazari M F & Porto C S 2012 17Beta-estradiol    signaling and regulation of proliferation and apoptosis of rat    Sertoli cells. Biology of Reproduction 86 108.-   Sato T, Kim H, Kakuta H & Iguchi T 2018 Effects of    2,3-Bis(4-hydroxyphenyl)-propionitrile on Induction of Polyovular    Follicles in the Mouse Ovary. In vivo 32 19-24.-   Shima Y, Miyabayashi K, Haraguchi S, Arakawa T, Otake H, Baba T,    Matsuzaki S, Shishido Y,-   Akiyama H, Tachibana T, et al. 2013 Contribution of Leydig and    Sertoli cells to testosterone production in mouse fetal testes.    Molecular endocrinology 27 63-73.-   Sittadjody S, Saul J M, Joo S, Yoo J J, Atala A & Opara E C 2013    Engineered multilayer ovarian tissue that secretes sex steroids and    peptide hormones in response to gonadotropins. Biomaterials 34    2412-2420.-   Tholpady S S, Katz A J & Ogle R C 2003 Mesenchymal stem cells from    rat visceral fat exhibit multipotential differentiation in vitro.    The anatomical record. Part A, Discoveries in molecular, cellular,    and evolutionary biology 272 398-402.-   Tuli R, Seghatoleslami M R, Tuli S, Wang M L, Hozack W J, Manner P    A, Danielson K G & Tuan R S 2003a A simple, high-yield method for    obtaining multipotential mesenchymal progenitor cells from    trabecular bone. Molecular biotechnology 23 37-49.-   Tuli R, Tuli S, Nandi S, Wang M L, Alexander P G, Haleem-Smith H,    Hozack W J, Manner P A, Danielson K G & Tuan R S 2003b    Characterization of multipotential mesenchymal progenitor cells    derived from human trabecular bone. Stem Cells 21 681-693.-   Whitby R J, Dixon S, Maloney P R, Delerive P, Goodwin B J, Parks D J    & Willson T M 2006 Identification of small molecule agonists of the    orphan nuclear receptors liver receptor homolog-1 and steroidogenic    factor-1. Journal of medicinal chemistry 49 6652-6655.-   Whitby R J, Stec J, Blind R D, Dixon S, Leesnitzer L M,    Orband-Miller L A, Williams S P, Willson T M, Xu R, Zuercher W J, et    al. 2011 Small molecule agonists of the orphan nuclear receptors    steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1    (LRH-1, NR5A2). Journal of medicinal chemistry 54 2266-2281.-   Wu L, Zhang A, Sun Y, Zhu X, Fan W, Lu X, Yang Q & Feng Y 2012 Sirt1    exerts anti-inflammatory effects and promotes steroidogenesis in    Leydig cells. Fertility and Sterility 98 194-199.-   Yang Y, Li Z, Wu X, Chen H, Xu W, Xiang Q, Zhang Q, Chen J, Ge R S,    Su Z, et al. 2017 Direct Reprogramming of Mouse Fibroblasts toward    Leydig-like Cells by Defined Factors. Stem cell reports 8 39-53.-   Yang Y, Su Z, Xu W, Luo J, Liang R, Xiang Q, Zhang Q, Ge R S & Huang    Y 2015 Directed mouse embryonic stem cells into leydig-like cells    rescue testosterone-deficient male rats in vivo. Stem cells and    development 24 459-470.-   Yazawa T, Imamichi Y, Miyamoto K, Khan M R, Uwada J, Umezawa A &    Taniguchi T 2016 Induction of steroidogenic cells from adult stem    cells and pluripotent stem cells [Review]. Endocrine journal 63    943-951.-   Yazawa T, Inanoka Y, Mizutani T, Kuribayashi M, Umezawa A & Miyamoto    K 2009 Liver receptor homolog-1 regulates the transcription of    steroidogenic enzymes and induces the differentiation of mesenchymal    stem cells into steroidogenic cells. Endocrinology 150 3885-3893.-   Yazawa T, Kawabe S, Inaoka Y, Okada R, Mizutani T, Imamichi Y, Ju Y,    Yamazaki Y, Usami Y, Kuribayashi M, et al. 2011 Differentiation of    mesenchymal stem cells and embryonic stem cells into steroidogenic    cells using steroidogenic factor-1 and liver receptor homolog-1.    Molecular and cellular endocrinology 336 127-132.-   Yazawa T, Mizutani T, Yamada K, Kawata H, Sekiguchi T, Yoshino M,    Kajitani T, Shou Z, Umezawa A & Miyamoto K 2006 Differentiation of    adult stem cells derived from bone marrow stroma into Leydig or    adrenocortical cells. Endocrinology 147 4104-4111.-   Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin, I I & Thomson    J A 2009 Human induced pluripotent stem cells free of vector and    transgene sequences. Science 324 797-801.-   Zhu M, Heydarkhan-Hagvall S, Hedrick M, Benhaim P & Zuk P 2013    Manual isolation of adipose-derived stem cells from human    lipoaspirates. Journal of visualized experiments: JoVE e50585.-   Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W, Katz A J, Benhaim P,    Lorenz H P & Hedrick M H 2001 Multilineage cells from human adipose    tissue: implications for cell-based therapies. Tissue engineering 7    211-228.

What is claimed is:
 1. A method of differentiating cells, the method comprising: incubating stem cells with a combination of SF-1 and WT1 agonists and inverse agonists to induce the cells to differentiate into gonadal hormone-producing cells.
 2. The method according to claim 1, wherein the hormone-producing cells are treated with a concentration of db-cAMP and/or gonadotropin (hCG, LH and/or FSH) to promote hormone synthesis and secretion from the cells.
 3. The method according to claim 1, wherein a therapeutic dose of hormone-producing cells is transplanted into a patient.
 4. The method according to claim 3, wherein the patient transplanted with hormone-producing cells is injected with a dose of gonadotropin (hCG, LH and/or FSH) or db-cAMP sufficient to promote further differentiation of transplanted hormone-producing cells.
 5. The method according to claim 3, wherein the patient transplanted with hormone-producing cells is injected with a dose of gonadotropin (hCG, LH and/or FSH) or db-cAMP sufficient to promote sex hormone synthesis and secretion.
 6. The method according to claim 5, wherein the patient transplanted with hormone-producing cells is injected with a dose of gonadotropin (hCG, LH and/or FSH) or db-cAMP sufficient to induce an increase in the circulating concentration of hormones to: maintain circulating concentration of hormones at or near physiological concentrations; and increase circulating concentration of hormones above physiological concentrations.
 7. The method according to claim 1, wherein the cells include one or more of: a type that regulates blood or tissue levels of hormones produced by gonads of the patient; a type that regulates the production of hormones produced by the gonads; a type that regulates the function of hormones produced by the gonads; a type that regulates the activity of hormones produced by the gonads; a type that regulates the blood or tissue levels of sex steroids, inhibins or follistatin; a type that regulates the production of sex steroids, inhibins or follistatin; a type that regulates the function of sex steroids, inhibins or follistatin; a type that regulates the activity of sex steroids, inhibins or follistatin; a type that increases the blood or tissue levels of sex steroids, inhibins or follistatin; a type that increases the production of sex steroids, inhibins or follistatin; a type that increases the function of sex steroids, inhibins or follistatin; a type that increases the activity of sex steroids, inhibins or follistatin; a type that regulates the blood or tissue levels of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the production of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the function of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the activity of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that decreases the blood or tissue levels of kisspeptin, GnRH, LH or FSH; a type that decreases the production of kisspeptin, GnRH, LH or FSH; a type that decreases the function of kisspeptin, GnRH, LH or FSH; a type that decreases the activity of kisspeptin, GnRH, LH or FSH; a type that decreases the blood or tissue levels of kisspeptin, GnRH, LH or FSH and increases the blood or tissue levels of sex steroids or inhibins; a type that decreases the production of kisspeptin, GnRH, LH or FSH and increases the production of sex steroids or inhibins; a type that decreases the function of kisspeptin, GnRH, LH or FSH and increases the function of sex steroids or inhibins; and a type that decreases the activity of kisspeptin, GnRH, LH or FSH and increases the activity of sex steroids or inhibins.
 8. The method according to claim 7 wherein the blood and/or tissue levels, production, function and activity, respectively, are regulated to be near the blood and/or tissue levels, production, function and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 9. The method according to claim 7 wherein the ratios of hormones in the HPG axis of the patient are maintained at or near a ratio occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 10. The method according to claim 9 wherein the sex steroids, inhibins and follistatin of the patient are regulated to be at the ratio near the blood and/or tissue levels, production, function, and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 11. The method according to claim 9 wherein the kisspeptin, neurokinin B, dynorphin. kit ligand, AMH, GnRH, LH, FSH and activins are regulated to be at the ratio near the blood and/or tissue levels, production, function, and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 12. The method according to claim 1, wherein the cells include one or more of: stem cells, stem cells that are differentiated cells or cells in other states for replacement in the brain, lung, eye, ear, tongue, nose, pancreas, liver, heart, bone, gonads, kidneys, spleen, pituitary, hypothalamus or other tissues; cells that are capable of differentiating into a hormone-producing cell type; cells that are capable of differentiating into one or more than one testicular cell type; cells that are capable of differentiating into one or more than one ovarian cell type; cells that are capable of differentiating into Leydig and Sertoli cells in the testes; cells that are capable of differentiating into granulosa and thecal cells in the ovary; cells that are capable of differentiating into one or more of spermatogonia, spermatocytes, spermatids, spermatozoon, Sertoli cells, myoid cells, Leydig cells, stromal cells, macrophage cells and/or epithelial cells in the testes; cells that are capable of differentiating into one or more of germ cells (oogonial stem cells), granulosa cells, cumulus cells, thecal cells, myoid cells, stromal cells, epithelial cells, macrophage cells and/or oocyte cells in the ovary; cells that include one or more stem cells and differentiated cell types; and cells that are autologous or allogeneic cells.
 13. A method of reducing endocrine dyscrasia (dyosis) in a patient, comprising: maintaining or rebalancing a hypothalamic-pituitary-gonadal (HPG) axis of the patient in need thereof by administering a therapeutically effective amount of the gonadal hormone-producing cells according to the method of claim 1
 14. The method according to claim 13 wherein the ratios of hormones in the HPG axis of the patient are maintained at or near a ratio occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 15. The method according to claim 14 wherein the sex steroids, inhibins and follistatin of the patient are regulated to be at the ratio near the blood and/or tissue levels, production, function, and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 16. The method according to claim 14 wherein the kisspeptin, neurokinin B, dynorphin. kit ligand, AMH GnRH LH, FSH and activins are regulated to be at the ratio near the blood and/or tissue levels, production, function, and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 17. A method of reducing rejection in a patient in need of a tissue-specific stem cell transplant, the method comprising: rebalancing a HPG axis of the patient in need thereof by administering a therapeutically effective amount of cells treated according to the method of claim 1; and administering stem cells that are tissue-specific to the patient.
 18. The method according to claim 17, wherein the cells include one or more of: a type that regulates the blood or tissue levels of hormones produced by the gonads; a type that regulates the production of hormones produced by the gonads; a type that regulates the function of hormones produced by the gonads; a type that regulates the activity of hormones produced by the gonads; a type that regulates the blood or tissue levels of sex steroids, inhibins or follistatin; a type that regulates the production of sex steroids, inhibins or follistatin; a type that regulates the function of sex steroids, inhibins or follistatin; a type that regulates the activity of sex steroids, inhibins or follistatin; a type that increases the blood or tissue levels of sex steroids, inhibins or follistatin; a type that increases the production of sex steroids, inhibins or follistatin; a type that increases the function of sex steroids, inhibins or follistatin; a type that increases the activity of sex steroids, inhibins or follistatin; a type that regulates the blood or tissue levels of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the production of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the function of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that regulates the activity of kisspeptin, neurokinin B, dynorphin, kit ligand, AMH, GnRH, LH, FSH or activins; a type that decreases the blood or tissue levels of kisspeptin, GnRH, LH or FSH; a type that decreases the production of kisspeptin, GnRH, LH or FSH; a type that decreases the function of kisspeptin, GnRH, LH or FSH; a type that decreases the activity of kisspeptin, GnRH, LH or FSH; a type that decreases the blood or tissue levels of kisspeptin, GnRH, LH or FSH and increases the blood or tissue levels of sex steroids or inhibins; a type that decreases the production of kisspeptin, GnRH, LH or FSH and increases the production of sex steroids or inhibins; a type that decreases the function of kisspeptin, GnRH, LH or FSH and increases the function of sex steroids or inhibins; and a type that decreases the activity of kisspeptin, GnRH, LH or FSH and increases the activity of sex steroids or inhibins.
 19. The method according to claim 18 wherein the blood and/or tissue levels, production, function and activity, respectively, are regulated to be near the blood and/or tissue levels, production, function and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 20. The method according to claim 18 wherein the ratios of hormones in the HPG axis of the patient are maintained at or near a ratio occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 21. The method according to claim 20 wherein the sex steroids, inhibins and follistatin of the patient are regulated to be at the ratio near the blood and/or tissue levels, production, function and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 22. The method according to claim 20 wherein the kisspeptin, neurokinin B, dynorphin. kit ligand, AMH, GnRH, LH, FSH and activins are regulated to be at the ratio near the blood and/or tissue levels, production, function and activity occurring during fetal life or at or near the time of greatest reproductive function of the patient.
 23. The method according to claim 17, wherein the cells include one or more of: stem cells, stem cells that are differentiated cells or cells in other states for replacement in the brain, lung, eye, ear, tongue, nose, pancreas, liver, heart, bone, gonads, kidneys, spleen, pituitary, hypothalamus or other tissues; cells that are capable of differentiating into a hormone-producing cell type; cells that are capable of differentiating into one or more than one testicular cell type; cells that are capable of differentiating into one or more than one ovarian cell type; cells that are capable of differentiating into Leydig and Sertoli cells in the testes; cells that are capable of differentiating into granulosa and thecal cells in the ovary; cells that are capable of differentiating into one or more of spermatogonia, spermatocytes, spermatids, spermatozoon, Sertoli cells, myoid cells, Leydig cells, stromal cells, macrophage cells and/or epithelial cells in the testes; cells that are capable of differentiating into one or more of germ cells (oogonial stem cells), granulosa cells, cumulus cells, thecal cells, stromal cells, epithelial cells, macrophage cells and/or oocyte cells in the ovary; cells that include one or more stem cells and differentiated cell types; and cells that are autologous or allogeneic cells.
 24. A method of treating a patient in need thereof, comprising administering to the patient a therapeutically effective amount of hormone-producing cells that have the capacity to maintain in balance, or rebalance the HPG axis of the patient, whereby the method is performed prophylactically or as a therapy, to maintain or restore, HPG axis balance, respectively.
 25. The method according to claim 24 further comprising: preventing or slowing dyosis in the patient by administering to the patient a therapeutically effective amount of hormone-producing cells that regulate or increase the production of hormones produced by the gonads.
 26. The method according to claim 24 wherein the therapeutically effective amount of hormone-producing cells is administered to the patient to allow a permissive environment, prior to the administration of stem cells into the bloodstream or other tissues, for the successful treatment of systemic or tissue-specific conditions and diseases.
 27. The method according to claim 24 wherein the therapeutically effective amount of hormone-producing cells is administered to the patient to allow a permissive environment, prior to the administration of stem cells into the bloodstream or other tissues, for the successful regeneration of tissues.
 28. The method according to claim 24 wherein the therapeutically effective amount of hormone-producing cells is administered to the patient to allow a permissive environment, prior to the administration of stem cells into the bloodstream or other tissues, for the successful restoration of function of tissues.
 29. The method according to claim 24 further comprising injecting stem cells into gonads or other tissues of the patient in response to at least partially rebalancing the HPG axis of the patient.
 30. The method according to claim 26 further comprising administration of stem cells into the bloodstream or other tissues of the patient after preventing, slowing or halting endocrine dyscrasia (dyosis) in the patient.
 31. The method according to claim 26 wherein the hormone-producing cells are administered into the bloodstream or other tissues of the patient for the treatment of at least one of: systemic conditions and diseases; tissue-specific conditions and diseases; for the regeneration of tissues; and for the restoration of function of tissues. 